Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene
Delivery of the CRISPR/Cas9 components to the plant cells is a key step in its application as a genome editing tool. Here, we compared Agrobacterium-mediated transformation and protoplast transfection with CRISPR/Cas9 components for potato genome editing. Two sgRNAs were designed to simultaneously d...
| Main Authors: | , , , , , , , , |
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| Format: | info:ar-repo/semantics/artículo |
| Language: | Inglés |
| Published: |
Springer Netherlands
2021
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| Subjects: | |
| Online Access: | http://hdl.handle.net/20.500.12123/9204 https://link.springer.com/article/10.1007%2Fs11240-020-02008-9 https://doi.org/10.1007/s11240-020-02008-9 |
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| author | Gonzalez, Matías Nicolás Massa, Gabriela Alejandra Anderson, Mariette Decima Oneto, Cecilia Andrea Turesson, Helle Olsson, Niklas Fält, Ann Sofie Hofvander, Per Feingold, Sergio Enrique |
| author_browse | Anderson, Mariette Decima Oneto, Cecilia Andrea Feingold, Sergio Enrique Fält, Ann Sofie Gonzalez, Matías Nicolás Hofvander, Per Massa, Gabriela Alejandra Olsson, Niklas Turesson, Helle |
| author_facet | Gonzalez, Matías Nicolás Massa, Gabriela Alejandra Anderson, Mariette Decima Oneto, Cecilia Andrea Turesson, Helle Olsson, Niklas Fält, Ann Sofie Hofvander, Per Feingold, Sergio Enrique |
| author_sort | Gonzalez, Matías Nicolás |
| collection | INTA Digital |
| description | Delivery of the CRISPR/Cas9 components to the plant cells is a key step in its application as a genome editing tool. Here, we compared Agrobacterium-mediated transformation and protoplast transfection with CRISPR/Cas9 components for potato genome editing. Two sgRNAs were designed to simultaneously direct Cas9 to the StPPO2 gene encoding for a tuber polyphenol oxidase (PPO). A binary vector (CR-PPO) was utilized for either Agrobacterium-mediated transformation or for transient expression in protoplasts, while ribonucleoprotein complexes (RNP-PPO) were additionally assayed in protoplasts. Editing efficiency varied, yielding 9.6%, 18.4% and 31.9% of edited lines from Agrobacterium-mediated transformation, RNP-PPO and CR-PPO transient expression in protoplasts, respectively. Furthermore, only the CR-PPO transient expression resulted in lines edited in all four StPPO2 alleles, observed in 46% of the edited lines and confirmed by tuber PPO activity and enzymatic browning analysis. Lines with on-target DNA insertions were found from all three approaches and characterized by sequencing. The dual-sgRNA strategy resulted in a low incidence of the targeted deletion, likely due to contrasting efficiencies between sgRNAs, that was partially evident in the in silico analysis. Our results demonstrate that gene editing efficiency in potato depends on the CRISPR/Cas9 delivery strategy and provide insights to consider when selecting the appropriate methodology. |
| format | info:ar-repo/semantics/artículo |
| id | INTA9204 |
| institution | Instituto Nacional de Tecnología Agropecuaria (INTA -Argentina) |
| language | Inglés |
| publishDate | 2021 |
| publishDateRange | 2021 |
| publishDateSort | 2021 |
| publisher | Springer Netherlands |
| publisherStr | Springer Netherlands |
| record_format | dspace |
| spelling | INTA92042021-04-28T10:58:07Z Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene Gonzalez, Matías Nicolás Massa, Gabriela Alejandra Anderson, Mariette Decima Oneto, Cecilia Andrea Turesson, Helle Olsson, Niklas Fält, Ann Sofie Hofvander, Per Feingold, Sergio Enrique Papa Edición de Genes Protoplastos Agrobacterium Tumefaciens Catecol Oxidasa Potatoes Gene Editing Protoplasts Catechol Oxidase Delivery of the CRISPR/Cas9 components to the plant cells is a key step in its application as a genome editing tool. Here, we compared Agrobacterium-mediated transformation and protoplast transfection with CRISPR/Cas9 components for potato genome editing. Two sgRNAs were designed to simultaneously direct Cas9 to the StPPO2 gene encoding for a tuber polyphenol oxidase (PPO). A binary vector (CR-PPO) was utilized for either Agrobacterium-mediated transformation or for transient expression in protoplasts, while ribonucleoprotein complexes (RNP-PPO) were additionally assayed in protoplasts. Editing efficiency varied, yielding 9.6%, 18.4% and 31.9% of edited lines from Agrobacterium-mediated transformation, RNP-PPO and CR-PPO transient expression in protoplasts, respectively. Furthermore, only the CR-PPO transient expression resulted in lines edited in all four StPPO2 alleles, observed in 46% of the edited lines and confirmed by tuber PPO activity and enzymatic browning analysis. Lines with on-target DNA insertions were found from all three approaches and characterized by sequencing. The dual-sgRNA strategy resulted in a low incidence of the targeted deletion, likely due to contrasting efficiencies between sgRNAs, that was partially evident in the in silico analysis. Our results demonstrate that gene editing efficiency in potato depends on the CRISPR/Cas9 delivery strategy and provide insights to consider when selecting the appropriate methodology. EEA Balcarce Fil: González, Matías Nicolás. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina. Fil: Massa, Gabriela Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina. Fil: Massa, Gabriela Alejandra. Universidad de Buenos Aires. Facultad de Agronomía; Argentina. Fil: Anderson, Mariette. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia. Fil: Décima Oneto, Cecilia Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina. Fil: Décima Oneto, Cecilia Andrea. Universidad de Buenos Aires. Facultad de Agronomía; Argentina. Fil: Turesson, Helle. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia. Fil: Storani, Leonardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina. Fil: Olsson, Niklas. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia. Fil: Fält , Ann Sofie. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia. Fil: Hofvander, Per. Swedish University of Agricultural Sciences. Department of Plant Breeding; Suecia. Fil: Feingold, Sergio Enrique. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina. 2021-04-28T10:45:48Z 2021-04-28T10:45:48Z 2021-01-25 info:ar-repo/semantics/artículo info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion http://hdl.handle.net/20.500.12123/9204 https://link.springer.com/article/10.1007%2Fs11240-020-02008-9 1573-5044 (online) 0167-6857 (print) https://doi.org/10.1007/s11240-020-02008-9 eng info:eu-repograntAgreement/INTA/PNBIO-1131024/AR./Desarrollo de sistemas alternativos de generación y utilización de variabilidad genética y su aplicación al mejoramiento de los cultivos. info:eu-repograntAgreement/INTA/2019-PE-E6-I115-001/2019-PE-E6-I115-001/AR./Edición génica, transgénesis y mutagénesis como generadores de nueva variabilidad en especies de interés agropecuario info:eu-repo/semantics/restrictedAccess application/pdf Springer Netherlands Plant Cell, Tissue and Organ Culture 145 : 291-305 (2021) |
| spellingShingle | Papa Edición de Genes Protoplastos Agrobacterium Tumefaciens Catecol Oxidasa Potatoes Gene Editing Protoplasts Catechol Oxidase Gonzalez, Matías Nicolás Massa, Gabriela Alejandra Anderson, Mariette Decima Oneto, Cecilia Andrea Turesson, Helle Olsson, Niklas Fält, Ann Sofie Hofvander, Per Feingold, Sergio Enrique Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene |
| title | Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene |
| title_full | Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene |
| title_fullStr | Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene |
| title_full_unstemmed | Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene |
| title_short | Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene |
| title_sort | comparative potato genome editing agrobacterium tumefaciens mediated transformation and protoplasts transfection delivery of crispr cas9 components directed to stppo2 gene |
| topic | Papa Edición de Genes Protoplastos Agrobacterium Tumefaciens Catecol Oxidasa Potatoes Gene Editing Protoplasts Catechol Oxidase |
| url | http://hdl.handle.net/20.500.12123/9204 https://link.springer.com/article/10.1007%2Fs11240-020-02008-9 https://doi.org/10.1007/s11240-020-02008-9 |
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